Glycobiology Advance Access originally published online on April 24, 2006
Glycobiology 2006 16(8):766-775; doi:10.1093/glycob/cwj120
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The structures of glycolipids isolated from the highly thermophilic bacterium Thermus thermophilus Samu-SA1
2 Dipartimento di Chimica Organica e Biochimica, Università degli Studi di Napoli Federico II, Via Cintia 4, I-80126 Napoli, Italy; 3 Division of Structural Biochemistry, Research Center Borstel and 4 Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Leibniz Center for Medicine and Biosciences, Parkallee 10, D-23845 Borstel, Germany; and 5 Istituto di Chimica Biomolecolare (ICB-CNR), via Campi Flegrei 34, 80078 Pozzuoli, Napoli, Italy
1 To whom correspondence should be addressed; e-mail: molinaro{at}unina.it
Received on March 28, 2006; revised on April 20, 2006; accepted on April 21, 2006
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Abstract |
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Thermophiles constitute a class of microorganisms able to grow at extremely elevated temperatures. Some of these species are classified as Gram-negative bacteria, because of the presence of an outer membrane in the cell envelope, which is located on the top of a thick murein layer. Unlike typical Gram-negative bacteria, the outer membranes of Thermus species are not composed of lipopolysaccharides but of peculiar glycolipids (GL), whose structures seem to be strictly involved in the adaptation to high temperatures. In this work, the complete structures of the major GL components from the cell envelope of the thermophilic bacterium Thermus thermophilus Samu-SA1 are presented. Protocols conventionally adopted for Gram-negative bacteria were used, and, for the first time, GL from Thermus were analyzed in their native form. Two GL and one phosphoglycolipid (PGL) were detected and characterized. The two GL, analyzed by nuclear magnetic resonance (NMR) spectroscopy and electrospray ionization Fourier transform ion cyclotron resonance (ESI FT-ICR) mass spectrometry, possessed the same tetrasaccharide structure linked to a glycerol unit or, alternatively, to a long-chain diol. Moreover, a PGL from Thermus was characterized for the first time, in which N-glyceroyl-heptadecaneamine was present. These molecules are chemically related to other GL from thermophile bacteria, in which they play a crucial role in the adaptation of cell membranes to heat.
Key words: ESI FT-MS / glycolipid / long-chain diol / NMR / Thermus thermophilus
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
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Bacteria belonging to the genus Thermus constitute a class of extremophile microorganisms with an optimum growth temperature between 70 and 75°C. It is generally believed that a crucial role in the thermal stability that these bacteria are endowed with is provided by the particular structure of their cell envelope. Actually, Thermus bacteria possess a thick murein layer (Quintela et al., 1995
, 1999) like Gram-positive bacteria, bearing at its outside an outer membrane, as in Gram-negative bacteria, and the molecular arrangement of both appears to be central in the adaptation process to high temperatures (Brock, 1978). In particular, several kinds of polar glycolipids (GL) have been isolated from the membranes of Thermus (Pask-Hughes and Shaw, 1982; Wait et al., 1997; Lu et al., 2004) and Meiothermus (Yang et al., 2004) bacteria, which share some common structural features, mainly concerning kind and sequence of monosaccharides present, and the nature of the lipid component. The microorganism investigated in this study, Thermus thermophilus Samu-SA1, is a thermohalophilic bacterium first isolated in the shallow marine hot springs on Mount Grillo, in Italy (Romano et al., 2004). Applying the protocol conventionally adopted for isolation of lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria, we recovered a lipid component that was further purified to yield a fraction composed of two GL (GL1 and GL2) and one phosphoglycolipid (PGL), which were fully characterized by Gas chromatography-mass spectroscopy (GC-MS), nuclear magnetic resonance (NMR) spectroscopy, and ESI Fourier transform mass spectrometry (FT-MS). This is the first time that a structure of a ‘PGL’ from Thermus is presented, and, interestingly, it is closely related to those of analogous molecules isolated from Deinococcus (Huang and Anderson, 1989, 1992), a genus highly resistant to environmental hazards, with whom Thermus is phylogenetically related (Hensel et al., 1986). This fact suggests that these molecules play a crucial role in the adaptation to heat. |
Results |
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Cell culture, extraction, and chemical characterization of GL
Cells of T. thermophilus Samu-SA1 were grown at 75°C using the standard TH medium and harvested, in the stationary growth phase, by centrifugation. Cell yield was
3.5 g/L wet weight (0.9 g/L dry weight).
Dried cells were washed with 1% aqueous phenol to remove exocellular polysaccharides and subsequently extracted utilizing the hot phenol/water procedure (Westphal and Jann, 1965), optimized for Gram-negative bacteria. By chemical analyses, we detected a lipid-containing fraction in the phenol extract, which was first purified by enzymatic digestion with RNase, DNase, and Proteinase K. Analysis by thin-layer chromatography (TLC) evidenced the presence of two fractions that were isolated by silica-gel, and the major fraction underwent complete chemical analysis.
Fatty acid analysis revealed the presence of iso- and anteisobranched C15:0 and C17:0 and of minor amounts of iso- and anteisobranched C14:0, C16:0, and C18:0. GC-MS analyses of the acetylated O-methyl glycosides showed the presence of glucose (Glc), galactose (Gal), and 2-deoxy-2-amino-glucose (GlcN), present in non-stoichiometric amounts. Three additional constituents, degraded by strong hydrochloric methanol treatment, could be detected after milder methanolysis and acetylation. These were identified, on the basis of the ion fragmentation in the MS spectra, as glycerol (Gro), glycerol-phosphate (GroP), and GlcNAcyl, in which the acyl moiety was C17:0. Three late-eluting components were also found, which were identified by EI-MS and CI-MS analyses after either acetylation or trimethylsililation, as octadecane-1,2-diol (OD), heptadecaneyl-amine (HA), and its N-glyceroyl derivative. Long-chain alkyldiols were previously found as components of polar GL from other Thermus species (Wait et al., 1997), where they are thought to replace acyl-glycerol in the GL structure, whereas the occurrence of glyceric acid and long-chain alkylamine was a new finding. Detection of glycosylation sites and ring size of the monosaccharides was achieved by methylation analysis, identifying 2-substituted-Glc, 2-substituted-Gal, 6-substituted-GlcN and terminal GlcN, all in pyranose form, and terminal galactofuranose (Galf).
NMR and ESI FT-MS analysis of the GL extract
The poor solubility of GL in many common solvent systems is one of the major obstacles in their structure determination. This problem is typically overcome by introducing chemical modifications, usually peracetylation, to improve the solubility and to allow the execution of experiments in solution, in particular, the recording of NMR spectra. In this case, optimal solubility for the sample was found in CHCl3 : CH3OH (1:2, by volume), and this allowed the recording of a full set of 1D and 2D-NMR experiments on the native GL.
ESI FT-MS, as well as NMR analyses, demonstrated the existence of a mixture. In particular, the charge-deconvoluted ESI FT-MS mass spectrum obtained in the positive-ion mode (Figure 1) revealed at least three different species with monoisotopic masses of 1176.857, 1409.979, and 1467.977 u, each one accompanied by a panel of correlated ions originating from different acyl chain length (
m = 14 u) and from sodium attachment (m = 22 u).
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In 1D and 2D NMR spectra, partially overlapping signals of diverse GL species were visible. In particular, in the region between 5.200 and 4.600 p.p.m. of the 1H-NMR spectrum signals for at least six anomeric protons (A–F in order of decreasing chemical shift) were visible (Figure 2). Moreover, in the same region of the spectrum, signals were present for at least four acylated carbinolic protons (G–L). In the aliphatic region between 2.500 and 0.500 p.p.m., signals were identified deriving from protons belonging to the amino-methylene group and to particularly deshielded aliphatic methylene groups (Tables I and II). By double quantum-filtered correlation spectroscopy (DQF-COSY) and total correlation spectroscopy (TOCSY) spectra, the full assignment of proton resonances of the components of the mixture was possible. Subsequently, 13C chemical shifts were assigned from observed correlations in the 1H,13C-heteronuclear single quantum coherence (HSQC) spectrum (Figure 2 and Table I). For the monosaccharides, the 3JH,H coupling constant values derived from the DQF-COSY spectrum established for each residue anomeric and relative configurations. All residues, except residue A, were pyranoses, as proven by the observed carbon chemical shift values and by the intra-residual long-range correlations between C-1/H-5 and H-1/C-5 that appeared for each residue in the 1H,13C-HMBC spectrum. The 1H signal at 5.028 p.p.m. (H-1A) correlated in the TOCSY spectrum to proton signals at 4.079, 4.015, 3.943, and 3.725 p.p.m. All of these signals showed, in the 1H,13C-HSQC spectrum, correlations with down-field shifted carbon signals, up to 84.4 p.p.m., suggesting the occurrence of a furanose ring, as confirmed by the intra-residual H-1/C-4 and C-1/H-4 correlations in the 1H,13C-HMBC spectrum. The 1JC,H anomeric coupling constant values were also observable in the 1H,13C-HMBC spectrum, because the pulse sequence used to carry out this experiment contained a low-pass filter, set to a value of 145 Hz. In this way, the rising of 1JC,H couplings for ring C-H could be selectively avoided, whereas it was still possible to recover the 1JC,H for anomeric protons and carbons (Bubb, 2003). The 1JC,H coupling constant value of 174.0 Hz for spin system A together with the 13C chemical shift of the anomeric carbon signal and the intra-residual nuclear Overhauser effect (NOE) correlations observed was diagnostic for the 
-anomeric configuration for A, thus identified as terminal -Galf. The 1JC,H anomeric coupling constant values of the other spin systems were similarly obtained. For spin system B (5.019 p.p.m.), -galacto configuration was identified, on the basis of the low 3JH,H values for H-1/H-2, H-3/H-4, and H-4/H-5 (3.4, 3.6, and <1 Hz, respectively). Typical down-field shift because of glycosylation was observed for the C-2 resonance, proving the substitution at O-2 of this residue. Thus, this residue was a 2-substituted -galactopyranose (2--Gal).
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Residues C and D (anomeric proton shifts at 4.956 and 4.900 p.p.m., respectively) were both identified as -gluco configured residues on the basis of the large 3JH,H values except for 3J1,2 (3.6 Hz). Gluco configuration was also confirmed by a 2D rotating frame Overhauser enhancement spectroscopy (ROESY) spectrum, in which dipolar correlations between H-2/H-4 and H-3/H-5 of both residues were observed. The finding that these two residues shared the same pattern of resonances except for H-1 supported the idea that they represented the same monosaccharide, in slightly different chemical and magnetic environments. Down-field shift because of glycosylation was observed for C-2 (80.2 p.p.m.), allowing the identification of a 2-substituted -glucopyranose (2--Glc).
For residue E, 3JH,H values and intra-residual NOE connectivity revealed the -gluco configuration. Moreover, the C-2 chemical shift value at 54.1 p.p.m., in the typical region of nitrogen-bearing carbons, implied the occurrence of 2-amino-2-deoxy-glucose. A typical proton resonance down-field shift because of acetylation was observable for H-2 (3.843 p.p.m.). Actually, this signal correlated in the 1H,13C-HMBC spectrum to a carbon at 173.4 p.p.m., which correlated to the methyl signal of the acetyl group at 1.998 p.p.m. Thus, this residue was identified as terminal 2-acetamido-2-deoxy--glucopyranose (t--GlcNAc).
Residue F (H-1 at 4.629 p.p.m.) was identified as GlcN on the basis of the C-2 resonance at 57.1 p.p.m. and of the high 3JH,H values, whereas the diagnostic NOE correlations observed between H-1, H-3, and H-5 and 1JC,H anomeric coupling constant value (165 Hz) unambiguously proved the ß-gluco configuration. Substitution occurred at O-6, as testified by the glycosylation shift for C-6 (67.1 p.p.m.). Also, an acylation shift was observed for H-2 (3.553 p.p.m.). In the HMBC spectrum, H-2 correlated to a carboxyl signal at 176.9 p.p.m., which correlated to a proton at 2.218 p.p.m., was identified as H to the carboxyl group of a fatty acid alkyl chain. This information proved the existence of the amide linkage with a fatty acid, namely, on the basis of previous chemical analysis, with C17:0. Thus, residue F was identified as 6-substituted 2-acylamido-ß-glucopyranose (6-ß-GlcNAcyl).
Of the anomeric region of the 1H-NMR spectrum, signals at 5.169, 5.176, 4.950, and 4.661 p.p.m. correlated in the 1H,13C-HSQC spectrum with carbon resonances at 70.4, 70.2, 73.2, and 76.4 p.p.m.. These signals allowed the identification of four spin systems, designed G, L, H, and I respectively. In particular, the signal at 5.169 p.p.m. (H-2G) showed correlations in the DQF-COSY with two diasterotopic methylene groups, at 4.140/4.381 (H-3a/H-3bG) and 3.631/3.710 (H-1a/H-1bG) p.p.m. On the basis of the chemical shifts for protons and carbon signals (Table I) and of the observed long-range correlations with carboxyl group resonances at 174.4 and 174.5 p.p.m., it was possible to identify residue G as a Gro moiety acylated at O-2 and O-3. The identified resonances for the acyl moieties are summarized in Table II. Full attribution was impossible because of the merging of methylene signals of the long fatty acid chains into one broad signal at 1.242 p.p.m.
Residue H was identified as the expected OD, on the basis of the observed scalar correlations (DQF-COSY) of the signal at 4.950 p.p.m. (H-2H), with one hydroxymethylene group (3.523/3.580 p.p.m., H-1a/H-1bH) and a methylene group in the aliphatic region (1.532 p.p.m.). Chemical shifts for the other protons and carbons of the alkyl chain were only partially distinguishable (Table I). In this case, acylation occurred at O-2, as proven by the long-range correlation with the carboxyl at 177.1 p.p.m.
Residue I was identified as the N-glyceroyl-HA unit. The signal at 4.661 p.p.m. (H-2I) showed a correlation with a methylene signal at 3.830 p.p.m., which gave a scalar correlation with a carboxyl carbon at 170.1 p.p.m. (C-1I, 1H,13C-HMBC). The same carbon signal also showed a correlation to the methylene signal at 3.152/3.237 p.p.m., which was identified as H-1'I and, thus, as the aminomethylene position, on the basis of the carbon chemical shift value (39.7 p.p.m.).
A second Gro unit was identified (L), starting from the resonances of H-2 at 5.176 p.p.m., from which two correlations with hydroxymethylene groups, resonating at 4.084/4.312 (H-3a/H-3bL) and 3.983 p.p.m. (H-1a/bL), could be identified. Correlations were observed for H-2 and H-3 with carbonyl groups (172.5 and 173.1 p.p.m.), suggesting acylation at O-2 and O-3, whereas the observed down-field displacement of the H-1 resonance, compared with the analogous position of residue G, implied phosphorylation at this site, in consistency with chemical analyses.
Connectivity between the identified spin systems were established on the basis of the inter-residual dipolar correlations detected in the 2D ROESY spectrum and scalar long-range correlations observed in the 1H,13C-HMBC spectrum. In particular, proton H-1A gave a strong NOE connectivity with H-2B and a scalar correlation with the carbon resonance at 76.7 p.p.m., suggesting the attachment at O-2 of the -Galp residue. This was linked at O-6 of residue F, namely the ß-GlcNAcyl residue, as confirmed by the occurrence of the dipolar correlation H-1B/H-6F and of the long-range correlation between H-1B and C-6B. A cross peak appeared in the ROESY spectrum between H-1F and a proton at 3.472 p.p.m., identified as H-2 of residues C and D. The information deriving from both ROESY and 1H,13C-HMBC spectra showed that residue C was linked to O-1 of residue G. In fact, a long-range correlation existed between H-1C and carbon at 66.6 p.p.m. (C-1G). Residue D appeared to be linked at O-1 of residue H, namely the OD.
These data can be summarized in the following structure:
-Galf-(1-2)--Gal-(1-6)-ß-GlcNAcyl-(1-2)--Glc-(1-1)-R
A B F C/D
with R = Gro (G) or OD (H), thus, the tetrasaccharide backbone is linked either to the glycero l unit G or to the OD H.
Residue E (t--GlcNAc) showed a NOE correlation with H-2I and an additional long-range correlation with C-2I that indicated the presence of the fragment -GlcNAc-(1–2)-N-glyceroyl-heptadecane-amine. These two spin systems, as well as the GroP L, did not show any dipolar or long-range correlation in NMR spectra with the structures so far identified, thus appearing as isolated fragments, likely belonging to a different molecular species within the blend.
Purification and complete characterization of GL1, GL2, and PGL from T. thermophilus Samu-SA1
To find out further structural details, we performed mild de-O-acylation with anhydrous hydrazine, and the product was purified on silica gel. Two fractions were obtained, composed by de-O-acylated GL (de-O-GL1 and de-O-GL2), and ESI FT-MS and 1D and 2D NMR analyses were performed on both the products. The approximate molar ratios of monosaccharides in both fractions were Gal:Glc:GlcN, 2:1:1.
NMR spectra recorded on the more abundant fraction (de-O-GL1) appeared rather simplified compared with that of the initial mixture (Figure 3). Nevertheless, the tetrasaccharide linked to the OD already identified was clearly recognizable. This affirmation was proven by the NOE correlation in the ROESY spectrum between H-1 of the 2--Glc residue and H-1H and by the observation of the scalar long-range inter-residual correlation in the 1H,13C-HMBC with C-1H. Final confirmation of the proposed structure was provided by ESI FT-MS of the de-O-GL1 (Figure 4). The charge-deconvoluted mass spectrum revealed an abundant peak with monoisotopic mass of 1185.760 u, which is in an excellent agreement with the calculated mass (1185.7597 u) of a molecule composed of OD-Hex3-HexN-C17:0. Comparison with the ESI-MS spectra of the mixture led to the identification of the first molecular species in the native form (GL1, Figure 5), where the molecule appeared to be O-acylated at O-2 of the OD residue by a C15:0, as testified by the molecular mass of 1409.979 u, differing from 1185.760 u by one C15:0 unit.
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The NMR data (Figure 3) of the second isolated fraction (de-O-GL2) showed high analogy with de-O-GL1, the only structural difference being the occurrence of a Gro moiety instead of the long-chain diol. Also in this case, the tetrasaccharide structure was recognized after complete 2D NMR analysis and resulted identical to the structure already found in de-O-GL1.
ESI-MS spectrum on de-O-GL2 (Figure 4) showed an intensive molecular peak with 991.519 u, in agreement to the proposed species Gro-Hex3-HexN-C17:0, with a minor peak at 829.465 u (m = 162 u), suggesting the presence of a minor compound lacking of a hexose residue. This minor form was not distinguishable in the NMR spectra. The two ion peaks visible in this spectrum at 1027.497 u (m = 36 u) and 1051.536 u (m = 60 u) were likely because of artifacts deriving from the purification procedure applied, since neither chemical analysis nor NMR investigation showed any species that could generate these molecular ions. Moreover, analogous mass differences are undetected in the intact mixture spectrum. The comparison with the ESI-MS spectrum of the blend allowed to relate de-O-GL2 to the species represented by the peak with a mass of 1467.977 u, identified as the molecular ion peak for the second GL (GL2, Figure 5). In fact, the mass difference of 476 u between the two peaks was consistent with the presence of one C15:0 and one C17:0 residues, suggesting that O-2 and O-3 of the Gro unit must be esterified by these two residues. In the same way, the species with a molecular mass of 1439.949 u was in account for a minor compound acylated by two C15:0 units.
It was not possible to find any product related to the phospholipid present in the native mixture, likely because of total degradation during de-O-acylation. Nevertheless, it was possible to compare the composition of the two GL so far identified with the results of the chemical and spectroscopical analyses performed on the native mixture (Table III), deducing the identity of the third expected molecule. This was identified as a phospholipid (PGL), containing in its structure the fragment -GlcNAc-(1–2)-N-glyceroyl-heptadecane-amine, previously identified, and acyl-glycerolphosphate. This hypothesis was supported by the detection, in the ESI spectrum executed on the mixture, of a peak with mass of 1176.857 u (Table IV), which was consistent with Gro-HexNAc-GroAN(CH2)16CH3-P-C17:0-C15:0 (PGL, Figure 5). From this mass spectrum, it was also possible to deduce that the Gro unit was esterified in the most abundant species by one C15:0 and one C17:0. It was also possible to detect a highly heterogeneous acylation pattern, indicated by the occurrence of ions with m = 14 u, a methylene group, suggesting variability in the chain length of fatty acid residues. Moreover, the family of ions centered at 1134.846 u (m = 42 u, acetyl group) indicated the presence of a small amount of a compound, where the GlcN residue was not acetylated. The absence of dipolar correlations between the glyceramide and the acyl-glycerol units, observed in the previous NMR data, was explained with the existence of a phospho-diester bridge connecting the two fragments, as confirmed by the detection of glycerol phosphate in chemical analysis. Such a structure was earlier found in bacteria belonging to the genus Deinococcus, a genus phylogenetically related to Thermus (Huang and Anderson, 1989, 1992). Figure 5 shows the structures of GL1, GL2, and PGL.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
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T. thermophilus belongs to Gram-negative bacteria. Only few microorganisms within this class, namely Sphingomonas capsulata (Kawahara et al., 2000
), Treponema denticola (Schultz et al., 1998), Fibrobacter succinogenes (Vinogradov et al., 2001), Borrelia burgdorferi (Ben-Menachem et al., 2003), and Chlroflexus aurantiacus (Meissner et al., 1988), have been found to be deficient of LPS. In a previous study, we ascertained the absence of these peculiar molecules in T. thermophilus HB8 (Silipo et al., 2004), finding indeed a membrane-associated polysaccharide with a non-repeating unit. In this study, we have continued this investigation on the cell envelope components of T. thermophilus and have isolated novel polar GL. Moreover, we suggest that the chemico-physical role played by LPS may be accomplished by these molecules that are provided with a lipid portion, which forms the main part of the outer leaflet of the membrane itself, and with a saccharide part directed toward the surrounding environment.
Polar GL and PGL have been found to be the major components of the cell wall of Thermus bacteria (Pask-Hughes and Shaw, 1982). The two GL reported in the present work share a tetrasaccharide structure, in which a long-chain alkyl diol may replace Gro. This feature was already found in Thermomicrobium roseum (Jackson et al., 1973; Perry, 1992) as well as in the membrane composition of Thermus scotoductus and Thermus filiformis (Wait et al., 1997).
In our opinion, the occurrence of long-chain alkyl diols within membrane GL structure can be seen as one of the Thermus adaptive responses to environmental stressors, due the greater chemical strength of the alkyl chain compared with the more labile ester bonds of acyl glycerol. Other thermophilic species also express uncommon structural features, as for example long-chain ethers in Aquifex pyrophilus (Huber et al., 1992) or ,
-dicarboxylic fatty acids in Thermotoga maritima (De Rosa et al., 1989), which may result in an increased resistance to heat, regarding either chemical stability or preservation of physical properties of the whole membrane. Nevertheless, it is presently not possible to state whether these chemical peculiarities represent a type of evolutionistic answer to environmental pressures. The same consideration can be done concerning the presence of the unusual phospholipid structure, that, interestingly, is closely related to compounds previously found in Deinococcus (Huang and Anderson, 1989, 1992). Deinococcus genus (Murray, 1986) and Thermus genus (da Costa and Rainey, 2001) belong to the same Phylum BIV but different Family (Deinococcaceae and Thermaceae, respectively). Deinococcus is, like Thermus, an extremophilic genus (Murray, 1986), endowed with high resistance to heat, desiccation, and radiation. From a biological point of view, it was proposed that the phospholipids isolated from Deinococcus could be chosen as taxonomic genus markers. The detection of the same kind of molecules in bacteria belonging to the genus Thermus contradicts this idea. Indeed, we can consider that Thermus and Deinococcus are phylogenetically related, and the finding in both of similar membrane components can open a debate on existing analogies in the metabolic patterns of the two genera. Moreover, the consideration that both species can survive in extreme environments once again suggests a crucial involvement of these membrane compounds in the increased resistance to stress factors respect to common mesophilic Gram-positive and Gram-negative bacteria.
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Materials and Methods |
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Cell growth
T. thermophilus Samu-SA1 (DSM 15284, ATTC BAA-951) was grown at 75°C in a 50 L fermenter (Biostat-D, Braun) with a mechanical agitation of 100 rpm and an aeration flux of 56%. Growth was followed turbidimetrically at 540 nm. The standard culture medium (TH medium) contained (gr/L) peptone (Oxoid) 8.0, yeast extract (Oxoid) 4.0, NaCl 2.0 at pH 7.0.
Cells were harvested in the stationary phase of growth by continuous-flow centrifugation on a Alfa Laval Model LAB 102 B-20 centrifuge. The pellet obtained was lyophilized.
Isolation and enzymatic purification of the GL component
Dried cells (3.5 g) were washed with 140 mL 1% aqueous phenol and kept at 4°C under stirring for 16 h. After centrifugation (8000 x g, 4°C, 1 h), the supernatant was removed, and the cells were extracted thrice with 60 mL of 45% aqueous phenol at 68°C, according to the conventional hot phenol-water procedure (Westphal and Jann, 1965). The phenol phase was diluted and dialyzed against water (3.500 kDa molecular weight cut-off). After dialysis, the extract was again centrifuged (8000 x g) and lyophilized, obtaining 210 mg of dried mass. The extract was digested with DNase, Rnase, and Proteinase K, dialyzed and freeze dried (49 mg, 1.4% of dry cells).
Sugar and fatty acid analyses
Monosaccharide analyses were realized by means of GC-MS of acetylated O-methyl glycosides derivatives, obtained after methanolysis (2 M HCl/MeOH, 85°C, 24 h) and acetylation with acetic anhydride in pyridine (85°C, 30 min). The absolute configuration of the monosaccharides was obtained according to the published method (Leontein and Lönngren, 1978).
Methylation analysis was performed using the modified Hakomori procedure (Hakomori, 1964) by Ciucanu and Kerek (1984). After chloroform/water extraction, the organic phase was evaporated and hydrolyzed with 4 M trifluoroacetic acid (100°C, 3 h), carbonyl reduced with NaBD4, acetylated with acetic anhydride : pyridine (1:1, v/v), and analyzed by GC-MS. For identification of OD, heptadecane-1-amine, and N-glyceroyl-heptadecane-1-amine, trimethylsililation was achieved treating the sample with bis(trimethylsilyl)trifluoroacetamide, 60°C, 30 min, followed by vacuum centrifugation.
Purification of crude and de-O-acylated glycoliopid extract
Chromatography for purification of GL was performed on Silica gel (Merk, 230–400 mesh) eluted with CHCl3 : MeOH (9:1 to 1:1, by volume), and the fraction collected were monitored by TLC, developed with CHCl3 : MeOH : H2O (65:25:4, by volume), and visualized with 0.1% Ce(SO4)2·4H2O, 5% (NH4)6Mo7O24·4H2O in 5.8% v/v H2SO4. The most abundant fraction eluted was treated with anhydrous hydrazine at 37°C for 30 min. The hydrazine was removed by evaporation under nitrogen and the product was again purified on Silica gel, eluting with CHCl3 up to CHCl3 : MeOH (1:1, by volume) followed by TLC performed as already described.
ESI FT-MS analysis of GL
FT-MS was performed in the negative- and positive-ion modes using an APEX II—Instrument (Bruker Daltonics, Billerica, MA) equipped with an actively shielded 7 T magnet and an (nano) ESI source. Mass spectra were acquired using standard experimental sequences as provided by the manufacturer. For the negative-ion spectra samples (10 ng µL–1) were dissolved in a 50:50:0.001 (v/v/v) mixture of 2-propanol, water, and triethylamine. For the positive-ion mode 50:50:0.03 (v/v/v) mixture of 2-propanol, water, 30 mM ammonium acetate adjusted with acetic acid to pH 4.5 was used. The samples were sprayed at a flow rate of 2 µL min–1. Capillary entrance voltage was set to 3.8 kV and drying gas temperature to 150°C. The spectra shown are charge-deconvoluted, using the XMASS-6.1 software, and mass numbers given refer to the monoisotopic molecular masses.
NMR spectroscopy
1D and 2D 1H-NMR spectra were recorded on a solution of 6 mg in 0.6 mL of MeOD : CDCl3 (2:1, by volume) at 25°C.1H- and 13C-NMR experiments were carried out using a Bruker DRX-600 equipped with a cryogenic probe. Spectra were calibrated with internal methanol (
H 3.300, C 49.5). ROESY was measured using data sets (t1 x t2) of 4096 ·x 1024 points, and 16 scans were acquired. A mixing time of 200 ms was used. Double quantum-filtered phase-sensitive COSY experiments were performed with 0.258-s acquisition time, using data sets of 4096 x 1024 points, and 64 scans were acquired. TOCSY experiments were performed with a spinlock time of 100 ms, using data sets (t1 x t2) of 4096 x 1024 points, and 16 scans were acquired. In all homonuclear experiments, the data matrix was zero-filled in the F1 dimension to give a matrix of 4096 x 2048 points and was resolution enhanced in both dimensions by a shifted sine-bell function before FT. Coupling constants were determined on a first-order basis from 2D phase-sensitive DQF-COSY (Piantini et al., 1982; Rance et al., 1983). HSQC and HMBC experiments were measured in the 1H-detected mode via single-quantum coherence with proton decoupling in the 13C domain, using data sets of 2048 x 512 points, and 64 scans were acquired for each t1 value. Experiments were carried out in the phase-sensitive mode according to the described method (States et al., 1982). A 60-ms delay was used for the evolution of long-range connectivity in the HMBC experiment. In all heteronuclear experiments, the data matrix was extended to 2048 x 1024 points using forward linear prediction extrapolation (De Beer and van Ormondt, 1992; Hoch and Stern, 1996).
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Conflict of Interest Statement |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and MethodsConflict of Interest StatementAcknowledgmentsReferences |
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We thank Hermann Moll and Regina Engel for technical assistance. This work was partly financed by MIUR, Rome (Progetti di Ricerca di Interesse Nazionale, 2004 to M.P.).
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Abbreviations |
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CI, chemical ionization; DQF-COSY, double quantum filtered correlation spectroscopy; EI, electron impact; ESI FT-MS, electrospray ionization Fourier transform mass spectrometry; Gal, galactose; Galf, galactofuranose; GC–MS, gas chromatography-mass spectroscopy; GL, glycolipids; Glc, glucose; GlcN, 2-deoxy-2-amino-glucose; Gro, glycerol; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single-quantum coherence; LPS, lipopolysaccharides; NOE, nuclear Overhauser effect; NMR, nuclear magnetic resonance; OD, octadecane-1,2-diol; PGL, Phosphoglycolipid; ROESY, rotating frame Overhauser enhancement spectroscopy; TLC, thin-layer chromatography; TOCSY, total correlation spectroscopy
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References |
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